Application Notes

Time Resolved Fluorescence in Unilamellar Vesicles

Introduction

The lipidic matrixes of biological membranes are lyotropic liquid crystals
in lamellar phase. As highly structured supramolecular phases, their dynamics
and structural properties, that not necessarily correlate, are all important
to characterize unambiguously a membrane and/or its changes. Many studies
concerning membrane's properties and processes make use of the fluidity
concept that has become an ill-defined term since it groups together
different parameters, related with diverse structural and dynamics properties.

Here we present the characterization of dynamic and structural properties of
large unilamellar vesicles (LUV) of the artificial lipid dipalmitoylphosphatidylcholine (DPPC)
using time resolved fluorescence techniques, including fluorescence intensity decay
and fluorescence anisotropy decay. As fluorescent probes DPH, and its derivatives,
TMA-DPH and TMAP-DPH, were used.

Materials and Methods

Sample Preparation

Large unilamellar vesicles(LUV) suspension were formed using an aqueous solution of
the non ionic detergent octaethylene glycol dodecyl ether (C12E8),
to solubilize DPPC. Vesicles spontaneously formed when the detergent was subsequently
removed by addition of hydrophobic Bio-Beads, and incubated at 4°C during 12 h.

Equipment

Time resolved fluorescence measurements were done using a K2 multifrequency phase and
modulation spectrofluorometer from ISS, Inc. The instrument was equipped wit
Glan-Thompson polarizers. For all probes, the exciting light was from a modulable
ISS 375 nm LED laser. The emission was measured through Schott KV-399 and WG-420
long band-pass filters. Intensity decay measurements were done with the exciting
light polarized parallel to the vertical laboratory axis, and the emission was
viewed through a polarizer oriented at 55°("magic angle" condition) and the phase
and modulation values were obtained at ten modulation frequencies in intervals
according to the fluorophore lifetime. For anisotropy decay determination,
differential phase angles and modulation ratios were obtained from parallel
and perpendicular oriented sinusoidal polarized emission at twelve modulation
frequencies between 2 and 200 MHz. Dimethyl-POPOP (1,4-bis[2]4-Methyl-5-phenyloxazoly benzene)
in ethanol (τ=1.45 ns) was used as a reference of intensity decay. 5 mm
path-length square quartz cuvettes were used. Sample temperature was controlled by
an external bath circulator.

Data Analysis

Time-resolved fluorescence data were analyzed using the ISS
Vinci analysis software package. The fitting function for the lifetime measurements
was the sum of a continuously distributed Lorentzian component and a discrete
component, fixed at 0.01 ns to account for scattered light. Anisotropy decay data
were fitted to a hindered rotator model of anisotropy decay, which is based on the
"wobble-in-cone" model, including a hindered rotation component (i.e. two rotational
correlation times, where the second rotational correlation time,
θ2 is fixed at a large value, 1 ms,
relative to the lifetime.

r(t) = (r0-r∞) exp(-t / θ1) + r∞

where r0 is the amplitude of the anisotropy decay at time 0,
θ1 is the fast rotational
correlation time of the anisotropy decay, and r∞ is the residual anisotropy at infinite time.
θ1 with the fluorophore
rotational rate R1 by R1 = 1/ 6θ1,
r∞ is related to S, the mean second rank-order parameter of
the fluorescent probe in the bilayer S = (r∞/r0)1/2)

Results

In this lamellar system our results show that as temperature increase,
the fluorescence lifetimes and rotational correlation time of the DPH
derivatives probes decrease. In this way, the fluorescence and rotational
dynamics has a proportional behavior with the temperature (Figs. 1).
Analysis of the data at one temperature through the different curves indicate
that the increase of cholesterol in the bilayer, elicit significant changes
in water penetration into the lamella. Dynamic quenching of probe fluorescence
by probe-water interactions has been proposed to reduce fluorescence lifetimes.
The migration of gauche-trans-gauche conformation along phospholipid acyl
chains and dynamic phospholipids head group fluctuations may permit the
transport of water into the bilayer. Indeed cholesterol has long been recognized
to decrease water penetration in lipids lamellas. In DPPC vesicles, at temperatures
corresponding to the liquid crystalline state (50ºC), lifetime trends of all probes
(Figs. 1 A to C, lifetime) indicate that cholesterol incorporation elicits an
overall hydration decrease at both lamellar depths. However, we have found that
in the gel state (25ºC), cholesterol incorporation induces an overall hydration
increase as sensed by the two deeper probes.

From anisotropy decay analysis we recovered: θ1
is the fast rotational correlation time of the anisotropy decay
(inversely related with the fluorophore rotational rate R1), and r∞
the residual anisotropy at infinite time, a dynamics and structural property respectively
(Figs. 1 A to C, θ1 and r∞).
Considering that the probe rotational rate reflects the orientational dynamics of
the surrounding phospholipids molecules, we suggest that cholesterol incorporation
elicits non monotonic changes in the orientational dynamics of the phospholipids,
with an initial decrease in the rotational rate at lower cholesterol content and
a subsequent increase at 40 mol %. These variations are larger in the gel state
(25ºC) and at the acyl chains deep region as sensed by DPH.

The changes in the residual anisotropy at infinite time r∞ related to the lipid
packing order, indicate a monotonous order increase with cholesterol
incorporation in the liquid crystalline state (50ºC), similar at all the bilayer
depths. However, at the gel state (25ºC), the results indicate slight monotonous
decreases at the two shallower depths and practically no effect at the deep hydrophobic region.